Researchers are now investigating new biomarkers for cerebral
ischemia that could bring greater objectivity to the diagnosis of ischemic stroke.

Emergency medicine physician Edward Jauch, MD, MS, and others discussed the new assays and their potential impact on ischemic stroke diagnosis at a session on stroke markers at the American Association for Clinical Chemistry annual meeting in July.

Ischemic stroke diagnosis is now largely a diagnosis of exclusion. Clinicians rely on patient history, clinical findings, imaging techniques, and laboratory tests to rule out conditions that mimic stroke. In fact, says Dr. Jauch, who is an assistant professor of emergency medicine and associate director for research at the University of Cincinnati College of Medicine, and a member of the regional stroke team, one of the first tests he orders when a patient presents with stroke symptoms is serum or finger-stick glucose because the symptoms of hypoglycemia often mimic the symptoms of stroke.

Ideally, however, Dr. Jauch and others would have a simple diagnostic test that would help determine whether a person is hypoglycemic, having a transient ischemic attack, or truly suffering from an ischemic stroke. "At present, there’s no widely available and rapid diagnostic test to aid in the diagnosis of cerebral ischemia," notes Daniel Laskowitz, MD, associate professor of neurology and neurobiology at Duke University Medical Center, Durham, NC. Speaking in the AACC session, Dr. Laskowitz called CT scans "exquisitely sensitive" for intracranial hemorrhage, and he says they can help exclude mass lesions. "But they’re simply not sensitive for the diagnosis of acute cerebral ischemia, which accounts for more than 80 percent of all strokes," he says.

Ischemic stroke occurs when an artery to the brain is blocked and the neurons are deprived of oxygen and glucose, leading to lack of cellular energy. In many cases, the blockage resolves itself over time, but if the artery remains blocked for more than a few minutes, neurons begin to die. Intracranial hemorrhage occurs either when a diseased blood vessel within the brain bursts, allowing blood to leak inside the brain (intracerebral hemorrhagic stroke) or when a blood vessel just outside the brain ruptures, filling the area of the skull surrounding the brain with blood (subarachnoid hemorrhagic stroke).

Some imaging technologies, such as diffusion-weighted magnetic resonance imaging studies, have shown promise in detecting brain damage caused by ischemic stroke, but these types of studies require advanced technology, special expertise, and timeliness—all of which are often unavailable or not possible when a patient appears with stroke symptoms in the emergency department of a community hospital at 2:00 AM. "MRI is not going to be the end-all answer for most patients with acute stroke, especially since about one-third of patients having a stroke can’t get an MRI for one reason or another," says Dr. Jauch, referring to those who are on respirators or have implanted devices, for example.

But Robert Christenson, PhD, director of the rapid response laboratories and professor of pathology at the University of Maryland School of Medicine, Baltimore, one of the session’s moderators, says it’s not enough to have a stroke marker that can aid in the diagnosis of cerebral ischemia. "We need early markers of cerebral ischemia," he says, "because if laboratorians can help clinicians diagnose ischemic stroke early, the clinicians have a better chance of helping patients."

Presenters at the AACC session on stroke markers noted that in some ways, diagnosing
ischemic stroke is similar to diagnosing acute myocardial infarction. In the
case of AMI, time is muscle, and in the case of stroke, time is neurons. "If
you look at the 4 million stroke survivors in the U.S. right now, and you look
at their outcomes after the initial event, only 10 percent have recovered to
baseline function," Dr. Jauch says. In contrast to AMI patients, who return
to their daily activities, "truly less than half of all stroke patients are
able to resume some form of normal lifestyle compared to what they had prior
to their stroke," he says. But outcomes can be improved if more people with
ischemic stroke are diagnosed as early as possible after the onset of symptoms.
Dr. Jauch described one study in monkeys that demonstrates irreversible paralysis
can be avoided if blood flow is restored to the brain within three hours (J
Neurosurg. 1981:54[6]:773-782).

Studies of thrombolytic use in stroke patients appear to bear this out. "If you get treated within 90 minutes with thrombolytics, you have an odds ratio greater than three of returning to baseline, whereas if you wait the full 180 minutes, your odds ratio is still above one, but we are losing the potential to salvage part of the brain," Dr. Jauch says. What’s at risk, he says, is the ischemic penumbra. "This is tissue that’s still viable, even though it may not be functioning well at the moment, and is potentially salvageable if you can restore blood flow quickly after symptom onset."

Currently, the standard of care should be the use of thrombolytics, Dr. Jauch says, though only four percent of stroke patients are treated with tissue plasminogen activator, the only drug now approved for use in stroke patients. Associated with this drug is a risk of hemorrhage. "If it’s administered in the setting of acute ischemic stroke where the blood vessel is blocked, six percent of those receiving the drug will bleed in their brain, and half of those will die," he says. On the upside, using t-PA in stroke victims results in 140 fewer deaths per thousand patients treated.

However, patients who have a large amount of brain tissue necrosis should not be given t-PA because there is a high risk of reperfusion hemorrhage, Dr. Laskowitz adds. "If we could actually get a blood test that could help us differentiate dead cells from injured cells, however, it would help us individualize therapy for patients, and determine who could benefit most from thrombolytic and other types of therapies," he says.

An ideal marker for ischemic stroke, Drs. Laskowitz and Jauch say, would be a single whole blood biomarker, a protein released from injured neurons, small enough to move easily across the blood-brain barrier, and elevated within three hours after onset of symptoms. "Unfortunately, even though there are many biomarkers that correlate with stroke, none of them are even close to being robustly sensitive and specific enough to be a stand-alone diagnostic marker for stroke," Dr. Laskowitz says.

Drs. Laskowitz, Jauch, and Christenson, along with Alan Wu,
PhD, chief of the clinical chemistry laboratory at San Francisco General Hospital
and professor of laboratory medicine at the University of California at San
Francisco, have been collaborating with Biosite Inc., San Diego, to validate
a panel of biomarkers that could aid in the diagnosis of cerebral ischemia.
"As much as we don’t want to have to go with a multi-marker approach, I think
that’s really the only alternative at this point," Dr. Wu says. "We’ve been
studying some of these panels in our medical centers, and early data indicate
that some of these combinations of markers have potential, not only to aid in
the diagnosis of cerebral ischemia, but for other purposes as well."

Though cerebrospinal fluid would probably be the best matrix to use when testing for cerebral ischemia, Dr. Wu says, it isn’t practical to perform lumbar punctures on patients who present to the emergency department with acute symptoms of stroke. Performing a lumbar puncture excludes stroke patients from thrombolytic therapies. Whole blood is easier to collect, independent of the setting. Therefore, if the test is placed on a point-of-care platform, like the Biosite Triage System, such a test could be performed in ambulances and other emergency medical system vehicles, as well as in the hospital’s ED. In addition, putting the test on a POC platform provides a turnaround time advantage, Dr. Wu notes.

What biomarkers have researchers examined in their quest for a stroke diagnostic? Dr. Laskowitz evaluated several potential stroke markers early in the process of developing Biosite’s six-marker stroke panel. "What we found after looking at a wide variety of markers is that four markers really stood out," he says. One of them was S100B, a marker of astrocyte activation and one of the most widely studied markers of cerebral ischemia. While S100B has its advantages, pilot studies have demonstrated that its release pattern is fairly slow. Says Dr. Wu: "At four hours after symptom onset, concentrations of S100B were still pretty low in a pilot study we performed, with a sensitivity of 12 percent. S100B levels began to increase only after about eight hours, so whether this will be an ideal marker for stroke remains to be seen." In the pilot, it took three days for the sensitivity of this marker to rise to a point where it might become sufficient for clinical use.

The other three markers initially found useful in the early Biosite study were matrix metalloproteinase 9 (MMP-9), vascular cell adhesion molecule (VCAM), and von Willebrand factor (vWF). "When we created a logistic regression model, in those patients presenting at less than six hours, MMP-9, von Willebrand factor, and VCAM provided a very accurate model, with a correlation index of .98 area under the ROC curve and a sensitivity and specificity of 90 percent," Dr. Laskowitz says. The study had one glaring limitation—it was enriched with normal controls, whereas in the clinical setting, the markers would be used to differentiate between ischemic stroke patients presenting to the ED and those with symptoms that mimic stroke.

In another study, Dr. Laskowitz and his colleagues again examined a panel of markers that included S100B and MMP-9, but D-dimer, C-reactive protein, and brain natriuretic peptide were added to the panel. The study, which included 130 patients presenting to the ED with symptoms of stroke, showed promising results. "In this study," Dr. Laskowitz says, "we found no difference between age, gender, or race between the strokes and the mimics, and demonstrated a sensitivity of 81 percent and a specificity of 70 percent." The positive and negative predictive values were about 90 percent in three hours, he says.

Other potential markers for cerebral ischemia are neuron-specific enolase (NSE), N-acetylaspartate (NAA), human serum carnosinase, and glial fibrillary acidic protein. "A lot of studies have focused on neuron-specific enolase," Dr. Wu says, "but I think that because it is a large molecule that may have trouble passing through the blood-brain barrier, it’s probably not going to be very useful as a stand-alone marker." Since NSE is already present in blood, measurements of NSE would have to exceed baseline concentrations in normal whole blood to be used as a diagnostic test for cerebral ischemia. "In addition, it takes at least eight hours before we can see a substantial release of NSE after stroke," Dr. Wu says.

Although little has been written about it, NAA is a unique marker that could be useful in diagnosing cerebral ischemia because it is the second most abundant free amino acid derivative in the mammalian brain after glutamate. "This marker is attractive,"Dr. Wu says, "because it is released very early in the process of cerebral ischemia, has a very low molecular weight that allows it to easily cross the blood-brain barrier, and is almost exclusively found in the neurons." He adds, "If we’re looking for a marker that’s very sensitive for the pathophysiology of stroke, perhaps even to the point of being able to detect the earliest stages of stroke, such as transient ischemic attack, this might be a very useful marker for early events."

In most studies, Dr. Wu says, NAA has been measured using MRI, and it could be interesting to compare the concentration of NAA in the blood of a person who may be suffering from cerebral ischemia with the concentration of NAA that appears on an MRI of the same person’s brain. "If you look for NAA in blood, you are looking for a higher concentration that’s been released, but if you are doing MRI studies, you are actually looking to see a depletion of NAA from neural tissue," he says. NAA clears the body rapidly when cerebral ischemia occurs, and because of that, high concentrations of it appear in the blood quickly after an ischemic stroke. "Because of its rapid clearance, however, it tends to return to normal levels within a couple of days, so you’d have to measure it early," Dr. Wu cautions.

Another potential marker of cerebral ischemia, human serum carnosinase, is a dipeptidase brain enzyme that hydrolizes carnosine, n-serine, and homocarnosine. A decreased level of human serum carnosinase shows up in stroke, and Dr. Wu hypothesizes that this may be due to the death of carnosinase-producing cells or to a down-regulation of the enzyme itself. He says, "Here you have a marker that goes in the opposite direction of NAA, and the hypothesis of some investigators is that perhaps you should combine one marker that increases with another that decreases and you’ll see a greater differentiation between the two."

Moving forward, Drs. Christenson and Wu warn that there are
likely to be speed bumps along the way. Says Dr. Christenson, "With a panel
of stroke markers, which is what Biosite is planning to submit to the FDA for
review, the calculation of an index from the six markers in the panel is going
to be the key part of the whole strategy." This raises a number of questions
for researchers and regulators.

First, he says, the Food and Drug Administration will review the pre-market approval application that Biosite plans to submit this year, and it’s unclear whether the agency will review all six markers as one device or as six separate tests. In addition, when assigning CPT codes for the panel, will the American Medical Association assign one CPT code, or will labs be required to bill them as separate tests? Since the six-marker panel will be unique, Medicare pricing for the device could also be problematic.

In addition, he says, "when you only report an index indicating that there
is a high, medium, or low likelihood of ischemic stroke, which Biosite will
likely do, all of the information provided by each of the individual markers
is in the background. Therefore, off-label indications may be very difficult
to do, especially if you only need the results of one marker for the off-label
use." There would be some merit in being able to report a result for each one
of the six Biosite tests, he says, though that may not be possible.

It’s likely that the six-panel marker will be able to aid in the diagnosis of neurologic conditions other than cerebral ischemia. Says Dr. Wu, "Almost all of these markers are released into the cerebrospinal fluid and blood following any type of cerebral injury, whether it be stroke, seizures, a subarachnoid hemorrhage, or any type of acute process." Any breakdown of the blood-brain barrier, which could occur in a variety of infectious diseases, would also be likely to affect the results the test reports out, and this must be studied. Using a panel approach makes it possible to select markers with unique temporal and physiologic features—for a more robust diagnostic tool.

At present, Biosite has enrolled 820 subjects presenting with acute focal neurologic deficit within 24 hours of symptom onset in its 14-center study, which includes U.S. and international sites. Blood is being drawn at enrollment, and at three, six, nine, 12, 24, and 48 hours after enrollment. The company’s goal was to register between 500 and 1,000 subjects.

For the moment, Biosite is keeping the identity of its six markers under wraps, but in a study published last year in Clinical Chemistry (2003;49: 1733-1739), the company shared some of its initial data. Says Dr. Wu, "Biosite underwent a proteomic discovery program in which it identified from 751 samples 50 putative markers of stroke that align the various pathophysiologic pathways, and in their initial trial identified five markers." These markers were S100B, B-type neurotrophic growth factor, von Willebrand factor, MMP-9, and monocyte chemotactic protein-1. According to the authors of the study, in a panel algorithm in which three or more marker values above their respective cutoffs were scored as positive, these five markers provided a sensitivity of 92 percent at 93 percent specificity for ischemic stroke samples taken within six hours from symptom onset. Laboratories will have to wait to see, however, which markers Biosite chooses for its final panel of six.

Once the FDA approves a stroke diagnostic, whether it’s manufactured by Biosite or another company, presenters at the AACC stroke session predict that it will quickly find uses not only in stroke diagnosis, but also in the cardiac arena. Patients who have cardiopulmonary bypass in the setting of coronary artery bypass grafting have a high risk for neurocognitive defects, and one to four percent have strokes, Dr. Laskowitz points out. "If we could use a diagnostic marker to determine which patients actually were very high risk for cerebral ischemia when they are on the table, this would be the ideal population in which to initiate a neuroprotective strategy," he says.

In addition, younger people who present with a bleeding aneurysm often experience vasospasm at a later point in time, and stroke diagnostics could present "an enormously important way to identify a patient at risk for vasospasm before they manifest a clinical deficit, when we could still treat them prophylactically," Dr. Laskowitz says.

Drs. Laskowitz and Jauch foresee great demand for an FDA-approved stroke diagnostic. "In a setting where you don’t have the expertise readily available," Dr. Jauch says, "I think it will be widely used to tease out some of the subtle strokes that are out there." For now, the key to better outcomes is better patient and clinician education, and the formation of better mechanisms to manage emergent stroke victims.

To that end, the Joint Commission on Accreditation of Healthcare Organizations has begun to certify stroke centers—and Dr. Jauch applauds their efforts.

"There has been a movement afoot for a couple of years to develop stroke centers like we have for trauma, and hospitals can apply to JCAHO to be designated as such," he says. The benchmark used now at the University of Cincinnati College of Medicine for JCAHO certification is this: Patients who arrive at the hospital with a stroke who are eligible for thrombolytic therapy must receive the drug within 60 minutes. "Our compliance rate is 80 percent," he says.

But the real goal is to boost the number of patients who are diagnosed accurately and treated appropriately with thrombolytics from the current four percent "to the potential 20 percent," he says, "reducing the mortality and morbidity of this devastating disease."